Anaplerotic reprogramming of endothelial cells in pulmonary hypertension.

项目总结 摘要肺动脉高压是一种肺血管细胞失控的致命性疾病。 扩散。内皮细胞(EC)过度生长会导致丛状病变的形成，这些病变 肺高压患者肺动脉内典型的癌样改变。增加的 肺组织中的氧化应激导致超氧化物与一氧化氮的反应。这将导致 生成高活性的过氧亚硝酸根(ONOO-)，进而生成硝基酪氨酸 蛋白质的修饰。PH患者的肺组织蛋白质硝化水平升高。 我们最近发表的工作表明，Akt中350个酪氨酸残基的硝化导致了 Akt信号的持续激活。Akt的激活是一种被广泛描述的细胞生存和应激 但Akt的激活受到调节性蛋白激酶/磷酸酶(PTEN，PI3K， PDK1和mTOR)来控制Akt的活动，并确保其仅暂时处于活动状态。病理性的 Akt通路的激活与多种癌症有关。我们假设在PH中， Akt的硝化作用增加eNOS向线粒体的移位，从而下调氧化 磷酸化和激活丙酮酸羧基酶(PC)，从而上调抗凝作用。这 激活失活导致内皮细胞病理性过度增殖， PH时形成丛状病变。此外，我们建议使用Akt-1来靶向病理性Akt信号。 与抗氧化剂(屏蔽肽)结合的仅影响硝化介导Akt的结合肽 激活。我们将在以下目标检验这些假说：目标1：阐明硝化作用的作用 (Y350)介导Akt在EC中激活失活。目标2：确定抑制是否 Akt硝化作用破坏了抗凋亡和增殖的EC表型的形成。目标3：实现 在SU5416/缺氧性PH模型中检测Akt屏蔽肽对EC过度增殖的影响。

PROJECT SUMMARY Pulmonary arterial hypertension (PH) is a fatal disease with uncontrolled pulmonary vascular cell proliferation. Excessive endothelial cell (EC) growth leads to the formation of plexiform lesions, which are a characteristic cancer-like change within pulmonary arteries of patients with PH. The increased oxidative stress in lungs of patients with PH leads to the reaction of superoxide with NO. This results in the formation of highly reactive peroxynitrite (ONOO-) which, in turn, can produce nitro-tyrosine modifications in proteins. Lung tissues from patients with PH have an increased level of protein nitration. Our recently published work indicates that the nitration of tyrosine 350 residue in Akt leads to the sustained activation of Akt signaling. The activation of Akt is a well described cell survival and stress response but Akt activation is tightly regulated by the regulatory kinases/phosphatases (PTEN, PI3K, PDK1 and mTOR) that control Akt activity and ensuring that it is only temporarily active. Pathological activation of the Akt pathway has been implicated in various cancers. We hypothesize that in PH, nitration of Akt increases translocation of eNOS to mitochondria, which downregulates oxidative phosphorylation and activates Pyruvate Carboxylase (PC), which upregulates anaplerosis. This activation of anaplerosis then leads to pathological hyper-proliferation of endothelial cells (EC) and plexiform lesion formation in PH. Further, we propose to target pathological Akt signaling using an Akt- binding peptide conjugated with an antioxidant (shielding peptide) that only affects nitration mediated Akt activation. We will test these hypotheses in the following Aims: AIM 1: To elucidate the role of nitration (Y350) mediated Akt activation in activation of anaplerosis in EC. AIM 2: To determine whether inhibition of Akt nitration disrupts formation of the apoptosis resistant and proliferative EC phenotype. AIM 3: To examine the effect of Akt shielding peptide on EC hyper-proliferation in the SU5416/hypoxia PH models.